The following is a brief description of nucleoside triphosphates. This summary is not meant to be complete but is provided only for understanding of the invention that follows. This summary is not an admission that all of the work described below is prior art to the claimed invention.
The synthesis of nucleoside triphosphates and their incorporation into nucleic acids using polymerase enzymes has greatly assisted in the advancement of nucleic acid research. The polymerase enzyme utilizes nucleoside triphosphates as precursor molecules to assembly oligonucleotides. Each nucleotide is attached by a phosphodiester bond formed through nucleophilic attack by the 3xe2x80x2 hydroxy group of the oligonucleotide""s last nucleotide onto the 5xe2x80x2 triphosphate of the next nucleotide. Nucleotides are incorporated one at a time into the oligonucleotide in a 5xe2x80x2 to 3xe2x80x2 direction. This process allows RNA to be produced and amplified from virtually any DNA or RNA templates.
Most natural polymerase enzymes incorporate standard nucleoside triphosphates into nucleic acid. For example, a DNA polymerase incorporates dATP, dTTP, dCTP, and dGTP into DNA and an RNA polymerase generally incorporates ATP, CTP, UTP, and GTP into RNA. There are however, certain polymerases that are capable of incorporating non-standard nucleoside triphosphates into nucleic acids (Joyce, 1997, PNAS 94, 1619-1622, Huang et al., Biochemistry 36, 8231-8242).
Before nucleosides can be incorporated into RNA transcripts using polymerase enzymes they must first be converted into nucleoside triphosphates which can be recognized by these enzymes. Phosphorylation of unblocked nucleosides by treatment with POCl3 and trialkyl phosphates was shown to yield nucleoside 5xe2x80x2-phosphorodichloridates (Yoshikawa et al., 1969, Bull. Chem. Soc.(Japan) 42, 3505). Adenosine or 2xe2x80x2-deoxyadenosine 5xe2x80x2-triphosphate was synthesized by adding an additional step consisting of treatment with excess tri-n-butylammonium pyrophosphate in DMF followed by hydrolysis (Ludwig, 1981, Acta Biochim. et Biophys. Acad. Sci. Hung. 16, 131-133).
Non-standard nucleoside triphosphates are not readily incorporated into RNA transcripts by traditional RNA polymerases. Mutations have been introduced into RNA polymerase to facilitate incorporation of deoxyribonucleotides into RNA (Sousa and Padilla, 1995, EMBO J. 14,4609-4621, Bonner et al., 1992, EMBO J. 11, 3767-3775, Bonner et al., 1994, J. Biol. Chem. 42, 25120-25128, Aurup et al., 1992, Biochemistry 31, 9636-9641).
McGee et al., International PCT publication No. WO 95/35102, describes the incorporation of 2xe2x80x2-NH2-NTP""s, 2xe2x80x2-F-NTP""s, and 2xe2x80x2-deoxy-2xe2x80x2-benzyloxyamino UTP into RNA using bacteriophage T7 polymerase.
Wieczorek et al., 1994, Bioorganic and Medicinal Chemistry Letters 4, 987-994, describes the incorporation of 7-deaza-adenosine triphosphate into an RNA transcript using bacteriophage T7 RNA polymerase.
Lin et al., 1994, Nucleic Acids Research 22, 5229-5234, reports the incorporation of 2xe2x80x2-NH2-CTP and 2xe2x80x2-NH2-UTP into RNA using bacteriophage T7 RNA polymerase and polyethylene glycol containing buffer. The article describes the use of the polymerase synthesized RNA for in vitro selection of aptamers to human neutrophil elastase (HNE).
This invention relates to novel nucleotide triphosphate (NTP) molecules, and their incorporation into nucleic acid molecules, including nucleic acid catalysts. The NTPs of the instant invention are distinct from other NTPs known in the art. The invention further relates to incorporation of these nucleoside triphosphates into oligonucleotides using an RNA polymerase; the invention further relates to novel transcription conditions for the incorporation of modified (non-standard) and unmodified NTP""s into nucleic acid molecules. Further, the invention relates to methods for synthesis of novel NTP""s.
In a first aspect, the invention features NTP""s having the formula triphosphate-OR, for example the following formula I: 
where R is any nucleoside; specifically the nucleosides 2xe2x80x2-O-methyl-2,6-diaminopurine riboside;2xe2x80x2-deoxy-2xe2x80x2-amino-2,6-diaminopurine riboside;2xe2x80x2-(N-alanyl)amino-2xe2x80x2-deoxy-uridine; 2xe2x80x2-(N-phenylalanyl)amino-2xe2x80x2-deoxy-uridine;2xe2x80x2-deoxy-2xe2x80x2-(N-xcex2-alanyl)amino; 2xe2x80x2-deoxy-2xe2x80x2-(lysiyl)amino uridine;2xe2x80x2-C-allyl uridine;2xe2x80x2-O-amino-uridine;2xe2x80x2-O-methylthiomethyl adenosine; 2xe2x80x2-O-methylthiomethyl cytidine; 2xe2x80x2-O-methylthiomethyl guanosine;2xe2x80x2-O-methylthiomethyl-uridine;2xe2x80x2-Deoxy-2xe2x80x2-(N-histidyl)amino uridine; 2xe2x80x2-deoxy-2xe2x80x2-amino-5-methyl cytidine;2xe2x80x2-(N-xcex2-carboxamidine-xcex2-alanyl)amino-2xe2x80x2-deoxy-uridine; 2xe2x80x2-deoxy-2xe2x80x2-(N-xcex2-alanyl)-guanosine; and 2xe2x80x2-O-amino-adenosine.
In a second aspect, the invention features a process for the synthesis of pyrimidine nucleotide triphosphate (such as UTP, 2xe2x80x2-O-MTM-UTP, dUTP and the like) including the steps of monophosphorylation where the pyrimidine nucleoside is contacted with a mixture having a phosphorylating agent (such as phosphorus oxychloride, phospho-tris-triazolides, phospho-tris-triimidazolides and the like), trialkyl phosphate (such as triethylphosphate or trimethylphosphate or the like) and dimethylaminopyridine (DMAP) under conditions suitable for the formation of pyrimidine monophosphate; and pyrophosphorylation where the pyrimidine monophosphate is contacted with a pyrophosphorylating reagent (such as tributylammonium pyrophosphate) under conditions suitable for the formation of pyrimidine triphosphates.
The term xe2x80x9cnucleotidexe2x80x9d as used herein is as recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1xe2x80x2 position of a sugar moiety. Nucleotides generally include a base, a sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No. WO 92/07065; Usman et al., International PCT Publication No. WO 93/15187; all hereby incorporated by reference herein). There are several examples of modified nucleic acid bases known in the art as recently summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183. some of the non-limiting examples of base modifications that can be introduced into nucleic acids without significantly effecting their catalytic activity include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azopyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine) and others (Burgin et al., 1996, Biochemistry, 35, 14090). By xe2x80x9cmodified basesxe2x80x9d in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1xe2x80x2 position or their equivalents; such bases may be used within the catalytic core of an enzymatic nucleic acid molecule and/or in the substrate-binding regions of such a molecule. Such modified nucleotides include dideoxynucleotides which have pharmaceutical utility well known in the art, as well as utility in basic molecular biology methods such as sequencing.
By xe2x80x9cunmodified nucleosidexe2x80x9d is meant one of the bases adenine, cytosine, guanine, uracil joined to the 1xe2x80x2 carbon of xcex2-D-ribo-furanose.
By xe2x80x9cmodified nucleosidexe2x80x9d is meant any nucleotide base which contains a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate.
By xe2x80x9cpyrimidinesxe2x80x9d is meant nucleotides comprising modified or unmodified derivatives of a six membered pyrimidine ring. An example of a pyrimidine is modified or unmodified uridine.
By xe2x80x9cnucleotide triphosphatexe2x80x9d of xe2x80x9cNTPxe2x80x9d is meant a nucleoside bound to three inorganic phosphate groups at the 5xe2x80x2 hydroxyl group of the modified or unmodified ribose or deoxyribose sugar where the 1xe2x80x2 position of the sugar may comprise a nucleic acid base or hydrogen. The triphosphate portion may be modified to include chemical moieties which do not destroy the functionality of the group (i.e., allow incorporation into an RNA molecule).
In another preferred embodiment, nucleoside triphosphates (NTP""s) of the instant invention are incorporated into an oligonucleotide using an RNA polymerase enzyme. RNA polymerases include but are not limited to mutated and wild type versions of bacteriophage T7, SP6, or T3 RNA polymerases.
In yet another preferred embodiment, the invention features a process for incorporating modified NTP""s into an oligonucleotide including the step of incubating a mixture having a DNA template, RNA polymerase, NTP, and an enhancer of modified NTP incorporation under conditions suitable for the incorporation of the modified NTP into the oligonucleotide.
By xe2x80x9cenhancer of modified NTP incorporationxe2x80x9d is meant a reagent which facilitates the incorporation of modified nucleotides into a nucleic acid transcript by an RNA polymerase. Such reagents include but are not limited to methanol; LiCl; polyethylene glycol (PEG); diethyl ether; propanol; methyl amine; ethanol and the like.
In another preferred embodiment, the modified nucleoside triphosphates can be incorporated by transcription into a nucleic acid molecules including enzymatic nucleic acid, antisense, 2-5A antisense chimera, oligonucleotides, triplex forming oligonucleotide (TFO), aptamers and the like (Stull et al., 1995 Pharmaceutical Res. 12, 465).
By xe2x80x9cantisensexe2x80x9d it is meant a non-enzymatic nucleic acid molecule that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993 Nature 365, 566) interactions and alters the activity of the target RNA (for a review see Stein and Cheng, 1993 Science 261, 1004; Agrawal et al., U.S. Pat. No. 5,591,721; Agrawal, U.S. Pat. No. 5,652,356).
By xe2x80x9c2-5A antisense chimeraxe2x80x9d it is meant, an antisense oligonucleotide containing a 5xe2x80x2 phosphorylated 2xe2x80x2-5xe2x80x2-linked adenylate residues. These chimeras bind to target RNA in a sequence-specific manner and activate a cellular 2-5A-dependent ribonuclease which, in turn, cleaves the target RNA (Torrence et al., 1993 Proc. Natl. Acad. Sci. USA 90, 1300).
By xe2x80x9ctriplex forming oligonucleotides (TFO)xe2x80x9d it is meant an oligonucleotide that can bind to a double-stranded DNA in a sequence-specific manner to form a triple-strand helix. Formation of such triple helix structure has been shown to inhibit transcription of the targeted gene (Duval-Valentin et al., 1992 Proc. Natl. Acad. Sci. USA 89, 504).
By xe2x80x9coligonucleotidexe2x80x9d as used herein is meant a molecule having two or more nucleotides. The polynucleotide can be single, double or multiple stranded and may have modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof.
By xe2x80x9cnucleic acid catalystxe2x80x9d is meant a nucleic acid molecule capable of catalyzing (altering the velocity and/or rate of) a variety of reactions including the ability to repeatedly cleave other separate nucleic acid molecules (endonuclease activity) in a nucleotide base sequence-specific manner. Such a molecule with endonuclease activity may have complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity that specifically cleaves RNA or DNA in that target. That is, the nucleic acid molecule with endonuclease activity is able to intramolecularly or intermolecularly cleave RNA or DNA and thereby inactivate a target RNA or DNA molecule. This complementarity functions to allow sufficient hybridization of the enzymatic RNA molecule to the target RNA or DNA to allow the cleavage to occur. 100% complementarity is preferred, but complementarity as low as 50-75% may also be useful in this invention. The nucleic acids may be modified at the base, sugar, and/or phosphate groups. The term enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, catalytic oligonucleotides, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme, finderon or DNA enzyme. All of these terminologies describe nucleic acid molecules with enzymatic activity. The specific enzymatic nucleic acid molecules described in the instant application are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target nucleic acid regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart a nucleic acid cleaving activity to the molecule.
By xe2x80x9cenzymatic portionxe2x80x9d or xe2x80x9ccatalytic domainxe2x80x9d is meant that portion/region of the ribozyme essential for cleavage of a nucleic acid substrate.
By xe2x80x9csubstrate binding armxe2x80x9d or xe2x80x9csubstrate binding domainxe2x80x9d is meant that portion/region of a ribozyme which is complementary to (i.e., able to base-pair with) a portion of its substrate. Generally, such complementarity is 100%, but can be less if desired. For example, as few as 10 bases out of 14 may be base-paired. That is, these arms contain sequences within a ribozyme which are intended to bring ribozyme and target together through complementary base-pairing interactions. The ribozyme of the invention may have binding arms that are contiguous or non-contiguous and may be varying lengths. The length of the binding arm(s) are preferably greater than or equal to four nucleotides; specifically 12-100 nucleotides; more specifically 14-24 nucleotides long. If a ribozyme with two binding arms are chosen, then the length of the binding arms are symmetrical (i.e., each of the binding arms is of the same length; e.g., six and six nucleotides or seven and seven nucleotides long) or asymmetrical (i.e., the binding arms are of different length; e.g., six and three nucleotides or three and six nucleotides long).
By xe2x80x9cnucleic acid moleculexe2x80x9d as used herein is meant a molecule having nucleotides. The nucleic acid can be single, double or multiple stranded and may comprise modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof. An example of a nucleic acid molecule according to the invention is a gene which encodes for macromolecule such as a protein.
By xe2x80x9ccomplementarityxe2x80x9d as used herein is meant a nucleic acid that can form hydrogen bond(s) with other nucleic acid sequence by either traditional Watson-Crick or other non-traditional types (for example, Hoogsteen type) of base-paired interactions.
In yet another preferred embodiment, the modified nucleoside triphosphates of the instant invention can be used for combinatorial chemistry or in vitro selection of nucleic acid molecules with novel function. Modified oligonucleotides can be enzymatically synthesized to generate libraries for screening.
In yet another preferred embodiment, the invention features a process for the incorporating a plurality of compounds of formula I.